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  • 1
    ISSN: 1573-5044
    Keywords: polypropylene membrane ; protoplast ; cell culture
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology
    Notes: Abstract A method of using a buoyant polypropylene membrane floated on liquid medium to culture asparagus cells and protoplasts was examined. Compared with direct culture in lquid medium the method using a polypropylene membrane was found to be superior for small-volume culture of cells at low density as well as for the culture of protoplasts.
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  • 2
    ISSN: 1573-5044
    Keywords: electropulsation ; Eucalyptus gunnii ; polyethylene glycol ; promoters ; protoplast ; transient gene expression
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology
    Notes: Abstract Conditions for optimal transient gene expression of reporter genes (chloramphenicol acetyl transferase and β-glucuronidase) by polyethylene glycol or electrical treatment of Eucalyptus gunnii protoplasts derived from callus or cell suspension cultures were investigated. The effciency of electropermeabilisation depended on several factors including electrical parameters, pH and the source of protoplasts. Polyethylene glycol mediated DNA uptake was highly stimulated by heat shock pretreatment and was also more efficient than the electrical treatment. For both treatments the nature of the promoter associated with the reporter gene was very important. A promoter corresponding to a protein synthesis elongation factor gene was more effective than the classical promoter of the 35 S transcript from cauliflower mosaic virus.
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  • 3
    ISSN: 1573-5044
    Keywords: alfalfa ; somatic embryogenesis ; protoplast ; cell suspensions
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology
    Notes: Abstract A system was established for achieving plant regeneration from mesophyll protoplasts and cotyledon-derived cell suspension cultures of alfalfa, Medicago sativa L. Peeled leaflets or cells from 6-day-old cell suspensions were incubated in an enzyme mixture containing 1% Driselase, 1% Rhozyme, 0.1% Cellulase and 72 gl-1 mannitol at pH 5.8 for 2–16 h to liberate protoplasts. A complex Kao medium supported cell division and colony formation, whereas a high auxin/low cytokinin treatment on Schenk and Hildebrandt medium followed by culture on growth regulator-free Blaydes or Linsmaier and Skoog medium resulted in somatic embryo formation. Of the three varieties tested. Citation, Answer and Regen S, the latter two produced embryos from which plants could be regenerated.
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  • 4
    ISSN: 1573-5044
    Keywords: protoplast ; grape ; Vitis ; vinifera ; tissue culture
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology
    Notes: Abstract A method of isolating grape mesophyll protoplasts was developed to facilitate the eventual use of genetic engineering techniques in this species. The effects of several factors influencing protoplast isolation could be evaluated quickly by using leaf disks 1 cm in diameter and known volumes of maceration and wash media. The best yields of mesophyll protoplasts were obtained using medium sized leaves of grapevines kept in the dark for 24 hours prior to maceration in 1% Cellulysin, 0.5% Macerase, 0.7 M mannitol, 5 ppm 2, 4 D, 0.1 ppm BAP, 1/10 strength Murashige and Skoog medium, and incubated at 22°C in cool-white fluorescent light (70–100 μE m-2 s-1) for 24 hours. Over 30×106 protoplasts per cm2 of leaf were produced using these conditions. This method of screening factors affecting protoplast isolation could be applicable to other species.
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  • 5
    ISSN: 1573-5044
    Keywords: Oryza sativa ; mutation ; protoclone ; protoplast ; totipotency
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology
    Notes: Abstract Protoplasts were isolated from callus derived from a single homozygous seed of Oryza sativa L. var. Norin 8. Thirty protoclones were randomly selected and these showed variation in regeneration frequency ranging from 0–87% with an average of 52%. The potential for regeneration of each protoclone as reflected in the regeneration frequency was analyzed five times over a period of 250 days and showed that the protoclones can be classified into three types, namely: protoclones with high regeneration frequency; protoclones with low regeneration frequency, both of which maintained their respective levels of regeneration potential; and protoclones with gradually decreasing regeneration frequency. Secondary protoclones established from protoplasts isolated from some of these protoclones and regenerated 2–3 times for a period of 120 days also showed further reduction in regeneration frequency. The polypeptide composition analyzed by two dimensional gel electrophoresis suggests the presence of specific polypeptides related to regeneration potential. Analysis of ploidy level based on plant morphology and pollen size suggests the predominance of tetraploids among the regenerated plants.
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  • 6
    ISSN: 1573-5044
    Keywords: plant regeneration ; protoplast ; suspension culture ; wheat
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology
    Notes: Abstract A fast-growing, small, granular, embryogenic callus was selected from primary calli induced from the Japanese wheat cultivar Nakasoushu and the Australian wheat cultivar Bodallin. Regenerable and fine suspension cultures were induced three to six months after liquid culture was initiated and were characterized by dense cytoplasm and active division. These suspension cultures routinely provided high yields of protoplasts with about 90% viability when incubated in a modified KMP (Kao and Michayluk, 1975) medium containing 1 mg l-1 2,4-D (2,4-dichlorophenoxyacetic acid), and 1 mg l-1 zeatin. Nakasoushu and Bodallin protoplasts divided at frequencies of 8.6% and 11.1%, respectively, in agarose-solidified media. When Nakasoushu protoplasts were cultured with effective nurse cells of sorghum and wheat, protoplast division increased to 16.9% and 12.6%, respectively. Plating efficiencies varied from 0.03% to 2.5%. After subculture, protocolonies yielded embryogenic calli and somatic embryos, from which green plants were eventually regenerated. Whole plants obtained from Nakasoushu protoplasts were fertile, demonstrating the first report of Japanese cultivars in wheat protoplast cultures.
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  • 7
    ISSN: 1573-5044
    Keywords: tomato ; protoplast ; cytoplasmic albino mutant ; regeneration
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology
    Notes: Abstract Mesophyll protoplasts from in vitro grown plants of a cytoplasmic albino mutant ofLycopersicon esculentum cv. Large Red Cherry were isolated with yields between 0.4 to 4.4 × 106 protoplasts per gram leaf tissue. Success in the culture of these protoplasts was dependent on embedding of the protoplasts in 100 µ1 agarose droplets 0.6% (w/v). A plating efficiency of 4.0% was obtained when the protoplasts were cultured in TM-2 medium with sucrose concentrations of 8.7 to 9.6% (w/v) resulting in an osmotic pressure of 432 to 469 mOsmol kg-1. After 14 days of protoplast culture, microcalli with a diameter of 3 mm were observed. After 3 weeks, macrocalli were obtained which were transferred to regeneration medium. Regeneration of shoot primordia, with a frequency of 19%, was obtained on TM-4 medium supplemented with 1% (w/v) sucrose. The first shoot primordia were visible 10 weeks after protoplast plating. For development of the shoot primordia into shoots it was necessary to increase the sucrose concentration to 6% (w/v). Eight out of eleven regenerants were diploid (2n = 2x = 24); the other three were tetraploid. Efficient regeneration of mesophyll albino protoplasts from tomato opens the way to select at the cellular level for the chloroplast transfers.
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  • 8
    ISSN: 1573-5044
    Keywords: Petunia hybrida ; Sorghum vulgare ; co-transformation ; electroporation ; inheritance ; phosphoenolpyruvate carboxylase ; protoplast
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology
    Notes: Abstract In previous work, transformedPetunia hybrida plants were obtained by direct gene transfer, using two different genes on separate plasmids (NPT II gene and a cDNA of PEPC from green sorghum leaves). In this study, we have analysed the sexual transmission of the acquired genes by genetic crossing analysis of 2 of the transgenic petunias. The ploïdies of the two clones were determined by flow cytometric analysis showing that one was 2n and the other 4n. Self and back crosses show that the kanamycin character was inherited as a single dominant trait, and that the two clones were heterozygotes for this character. Therefore, the 4n clone probably arises from an endoploidization followed by a transformation event. Southern blot analyses show that all of the resistant progenies which were analysed harboured the kanamycin gene, and expressed the phosphorylation activity in vitro. The DNA of several progenies were also tested for the presence of co-transformed PEPC cDNA sequence. All of the kanamycin-resistant progenies tested contained the second coding sequence, indicating that the two foreign genes might be genetically co-inherited in the transgenic plants. The way in which the two genes are integrated into the genome is discussed.
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  • 9
    ISSN: 1573-5044
    Keywords: electroporation ; pea ; Pisum sativum L. ; protoplast ; transformation
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology
    Notes: Abstract Protoplasts from two different pea cultivars, Belman and Filby, were stably transformed by direct gene transfer using electroporation. Transgenic calli could be obtained after selection, when hygromycin resistance was used as the selective trait introduced into the protoplasts, while no transformants were obtained when kanamycin resistance was used as selective marker in either of the two pea cultivars tested. The effect of the field strength on survival and division rates of the protoplasts was studied. Two different culture systems and osmotica were compared for induction of sustained divisions in and regeneration of transgenic callus from the protoplasts. The choice of the culture system had a considerable effect on the initial division frequency of the treated protoplasts, as well as on the later growth of the colonies. Transformation efficiency was monitored by histochemical GUS assay, and the transgenic nature of the calli selected for resistance against antibiotics was confirmed by DNA analysis.
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  • 10
    ISSN: 1573-5044
    Keywords: Brassica oleracea ; protoplast ; plating efficiency ; plant regeneration ; ‘rapid cycling’ ; var. Botrytis ; var. gemmifera
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology
    Notes: Abstract The results are reported for the first time on successful plant regeneration from mesophyll-derived protoplasts of ‘rapid cycling’ B. oleracea. Comparative data were also presented on plant regeneration from mesophyll-derived protoplasts of two other varieties namely var. botrytis and var. gemmifera. It was found that a modified Pelletier (Pelletier et al. 1983) protocol is highly beneficial for protoplast culture and plant regeneration from mesophyll-derived protoplasts. The plating efficiency of B. oleracea ‘rapid cycling’ protoplasts was, in the best combination of isolation method, culture technique and culture media 4.5%±0.4% and the plant regeneration frequency approximately 15%. Plant regeneration was further improved by transferring the calli from the D medium of Pelletier to a callus growth medium (MS11) and subsequently to the K3 regeneration medium of Glimelius (Glimelius 1984). Various factors influencing plating efficiency and plant regeneration from mesophyll protoplasts of B. oleracea are discussed.
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